PROJECT SUMMARY: Immunity to viral vectors precludes repeat dosing and decreases efficacy of vector-based gene therapies, which have otherwise shown promise in treating a range of genetic diseases. For increased efficacious imple- mentation of viral vector gene therapies, there is a critical need to address immunity to the therapeutic vector and transgene in a potent, antigen-specific manner. Without an appropriate solution to the immunogenicity of vector-based gene therapies, the clinical efficacy of many promising gene therapies will likely remain low. The long-term goal of SQZ Biotech is to develop a cell therapy to induce immune tolerance to viral vectors and their transgenes in patients receiving gene therapy. Using the patented Cell Squeeze system, materials like proteins and peptides can be reproducibly delivered to red blood cells (RBCs). Administration of SQZ-engineered RBCs induces tolerance to the delivered materials through endogenous, tolerogenic clearance of RBCs during eryp- tosis in the liver and spleen. For this Phase I SBIR proposal, the overall objectives are to simultaneously induce tolerance to the viral capsid and transgene product and to demonstrate that the SQZ-RBC approach induces tolerance to multiple AAV serotypes. The central hypothesis is that SQZ-RBCs will prevent in vivo immunity to capsid protein and transgene and that tolerance induced to AAV2 capsid protein will prevent immunity to other serotypes. Our hypothesis is based on preliminary data demonstrating that treating mice with RBCs SQZ?d with the immunodominant epitope of AAV2 capsid prevented reactivity to capsid and that the capsid sequence is highly conserved across serotypes. The rationale is that simultaneously inducing tolerance to AAV capsid and transgene will provide strong support for developing a clinically relevant therapy as immunity can develop to either or both components. In addition to broadening the applicability of the product, demonstrating that toleriza- tion to AAV2 induces tolerance across serotypes will enable quicker formation of strategic partnerships with gene therapy companies. To induce tolerance simultaneously to AAV capsid protein and transgene product in Aim 1, mice dosed multiple times with luciferase-expressing AAV will be treated prophylactically or therapeutically with SQZ-RBCs encapsulating luciferase and capsid protein. Serum levels of luciferase and T cell responsiveness to capsid and transgene will be measured to assess tolerization efficacy. To tolerize to multiple serotypes using AAV2 capsid in Aim 2, mice dosed multiple times with luciferase-expressing AAV5 or AAV8 will be treated prophylactically or therapeutically with RBCs SQZ?d with AAV2. Serum levels of luciferase and T cell respon- siveness to AAV2 capsid and AAV5 or AAV8 capsid will be measured to assess efficacy. Successful completion of these aims will establish a strong foundation for further preclinical development and the formulation of a human RBC product for future clinical trials. The proposed research would significantly progress development of a novel tolerance approach that enables better and broader application of gene therapies, which has the potential to significantly change the course of genetic disease treatment.